Emmanuel Amabebe ^1* Nadia Ikumi ² Ally Oosthuizen ² Priya Soma-Pillay ³ Mushi Matjila ² Dilly O. Anumba ^1*

• ¹ University of Sheffield, Sheffield, United Kingdom
• ² University of Cape Town, Cape Town, South Africa
• ³ University of Pretoria, Pretoria, South Africa

To explore relationships between markers of spontaneous preterm birth (sPTB), we: a) characterised the cervicovaginal fluid (CVF) cytokine profiles of pregnant South African women at risk of PTB; b) determined CVF matrix-metalloproteinase-9 (MMP-9) and its regulator tissue inhibitor of metalloproteinase-1 (TIMP-1); and c) explored the predictive potential of these markers for sPTB. The concentrations of 10 inflammatory cytokines, and MMP-9 and TIMP-1 were determined by ELISA in CVF samples from 47 non-labouring women at three gestational time points (GTPs): GTP1 (20-22 weeks, n=37), GTP2 (26-28 weeks, n=40), and GTP3 (34-36 weeks, n=29), and analysed for changes in protein concentrations and predictive capacities (area under the ROC curve (AUC) and 95% confidence interval (CI)) for sPTB. Eleven (GTP1), thirteen (GTP2) and six (GTP3) women delivered preterm within 85.3±25.9, 51.3±15.3 and 11.8±7.5 days after assessment respectively. At GTP1, IL-8 was higher (4-fold, p=0.02), while GM-CSF was lower (~1.4-fold, p=0.03) in the preterm compared to term women with an average AUC=0.73. At GTP2, IL-1β (18-fold, p<0.0001), IL-8 (4-fold, p=0.03), MMP-9 (17-fold, p=0.0007), MMP-9/TIMP-1 ratio (9-fold, p=0.004) and MMP-9/GM-CSF ratio (87-fold, p=0.005) were higher in preterm compared to term women with an average AUC=0.80. By contrast, IL-10 was associated with term delivery with an AUC (95%CI)=0.75(0.55-0.90). At GTP3, IL-1β (58-fold, p=0.0003), IL-8 (12-fold, p=0.002), MMP-9 (296-fold, p=0.03) and TIMP-1 (35-fold, p=0.01) were higher in preterm compared to term women with an average AUC=0.85. Elevated IL-1β was associated with delivery within 14 days of assessment with AUC=0.85 (0.67-0.96). Overall, elevated MMP-9 at GTP3 had the highest (13.3) positive likelihood ratio for distinguishing women at risk of sPTB. Lastly, a positive correlation between MMP-9 and TIMP-1 at all GTPs (⍴≥0.61, p<0.01) for women delivering at term was only observed at GTP1 for those who delivered preterm (⍴=0.70, p<0.03). In this cohort, sPTB is associated with gestation-dependent increase in pro-inflammatory cytokines, decreased IL-10 and GM-CSF, and dysregulated MMP-9-TIMP-1 interaction. Levels of cytokine (especially IL-1β) and ECM remodelling proteins rise significantly in the final two weeks before the onset of labour when sPTB is imminent. The signalling mechanisms for these ECM remodelling observations remain to be elucidated.